This research aims at understanding pyridine nucleotide turnover and regulation. A major effort in the next grant period will be to understand the nuclear metabolism of NAD in eukaryotes, specifically as a substrate for poly (ADP-ribosylation). A biochemical characterization of DNA stimulatory and binding sites, and poly ADP-ribose attachment sites is planned. In addition, a program of microinjection of labeled purified enzymes into living mammalian cells in culture will be carried out. A combined biochemical and genetic approach will also be continued in the bacterium Salmonella typhimurium. All remaining undefined genes involved in pyridine nucleotide will be identified and mutants at these loci obtained and characterized. Mutants in genes of the pyridine nucleotide cycle will be used to develop a convenient method to assay DNA ligation in vivo. The regulation of pyridine nucleotide pathways for biosynthesis and turnover will be investigated; the initial approach will be to construct Mu-lac fusions in all known genes.
